Folate polyglutamate synthesis is being studied in rat liver and in mammalian tumor cells propagated in vitro. The regulation of this enzyme activity by the end products of folate metabolism, as well as development of inhibitors of the enzyme are being investigated. Work is continuing on the characterization of homogenous thymidylate synthetase obtained by affinity chromatography. The machanism of the methotrexate-fluorouracil synergism that we are pursuing involves the pile-up of dihydrofolate polyglutamates as a consequence of methotrexate inhibition of dihydrofolate reductase, and on enhancement of FdUMP binding to thymidylate synthetase by these polyglutamates. Work continues on the molecular biology of dihydrofolate reductase. Multiple forms of dihydrofolate reductase messenger RNA have been identified by "Northern" blot analysis, and their function and synthesis is being examined. Genomic dihydrofolate reuctase is being cloned in gamma phage.